Week 14:Virtual Screening was continued and the final presentation was prepared.
Week 15: Virtual Screening (Small Libraries) and Controls were finished. The image for the positive and negative controls is shown below. The cloning attempt has been started.
Score
S(PLP)
S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
Ligand Name
Rank
Yes/No
68.38
-66.96
3.76
0
0
76.7
3.43
80.02
378
1
Yes
66.43
-51.7
6.64
0
0
55.7
1.76
2.31
783559
2
Yes
64.37
-50.87
6.73
0
0
0
1.99
2.29
894678
3
No
64.13
-65.12
2.39
0
0
0.08
2.61
1.47
9273753
4
Yes
62.84
-50
6.61
0
0
0
1.94
2.27
4588567
5
No
61.96
-46.75
6.71
0
0
85.47
2.72
78.72
3151007
6
Yes
61.11
-57.4
4.67
0
0
0
2.16
0.19
6934454
7
Yes
57.69
-49.05
6.49
0
0
0
1.84
2.25
9047838
8
Yes
55.53
-64.1
1.21
0
0
21.2
1.15
0.3
8345128
9
No
51.81
-42.21
4.61
0
0
0
1.68
2.35
3798080
10
Yes
48.63
-31.48
6.88
0
0
88.94
0.62
0
Aspirin(2244)
>10
Yes
45.56
-21.41
5.41
0
0
25.6
1.23
0.39
3123405
>10
Yes
Week 8: In Week 8, the Peer Review for the Mid-Semester Report was done. In the wet lab, the Oligo mix was prepared with 12 primers from the "C" container (Lanes G12, H1-H11).
Week 9: Secondary PCR was done and the agarose gel was run with 1 microliter of ethidium bromide. When the gel was run with both primary and secondary products, the ladder slid off the gel as did the PCR products. The gel was cracked in the lower half of the gel. It was accidentially thrown away along with the other gel from primary PCR and so, no picture was taken of the gel.
Week 10: Materials and Methods for Cloning was done. Since the first attempt at secondary PCR was a failure, another secondary PCR was done. However, the primary PCR products had been moved and the microcentrifuge tube had been bent and emptied of its products. So, primary PCR had to be done again and the results are coming in Week 11.
Week 3: The pNIC-Bsa4 culture was started. They were incubated overnight and spun down the next morning. The culture had contamination (pictures upcoming soon) of what appeared to be fungi so Aakash's pNIC culture was used for this procedure. It appeared the kanomycin was not effective for this process. The pNIC pellets (2) were placed in the -20 degree Celsius freezer for further use. Each pellet represented 40 mL worth of plasmid-containing bacterial cells.
Week 4: RE digest was done using EcoR1 and PvuII as the restriction enzymes. The inactivation temperature for PvuII was unknown and the inactivation temperature for EcoR1 was 60 degrees Celsius so 80 degrees Celsius was used for the heat block. In the 1.7 mL centrifuge tube, there was 18.95 microliters of plasmid, 2.5 microliters of 10X Enzyme buffer, 2.55 microliters of DDW to 24 microliters, 1 microliter of RE for each of the 3 tubes (the third tube had 0.5 microliters of PvuII and 0.5 microliters of EcoR1), with a total volume of 25 microliters. The gel picture is coming soon!
Later that week, Midi prep was done. Since all of the measurements in the protocol are based off of 20 mL of plasmid-containing bacterial cells and each pellet in this lab contained 40 mL of plasmid-containing bacterial cells, all the measurements were doubled from what they were in the protocol. The cells were resuspended in 1.6 mL of MX1 buffer, then 2 mL of MX2 buffer was added. 2.8 mL of chilled MX3 buffer was added, 6 mL of Wash buffer was used, and 2 mL of elution buffer was used in this lab. Speedvac was used instead of the 30-60 incubator when the membrane was dried. The elute was collected and stored for later use (Picture also coming soon!).
Week 1&2
Week 1: LB Agar solution was made. Week 2: LB+Kan solution was made. DNA sequencing and transformation took place. The forward sequence (Figure 2) did not work as planned, with many Ns dispersed throughout. The reverse sequence (Figure 1) was much better, with fewer N nucleotides dispersed in the middle of the sequence.
Figure 1: The Reverse Sequence of DNA (top)
Figure 2: The Forward Sequence of DNA (bottom)
Week 15: Virtual Screening (Small Libraries) and Controls were finished. The image for the positive and negative controls is shown below. The cloning attempt has been started.
Week 8: In Week 8, the Peer Review for the Mid-Semester Report was done. In the wet lab, the Oligo mix was prepared with 12 primers from the "C" container (Lanes G12, H1-H11).
Week 9: Secondary PCR was done and the agarose gel was run with 1 microliter of ethidium bromide. When the gel was run with both primary and secondary products, the ladder slid off the gel as did the PCR products. The gel was cracked in the lower half of the gel. It was accidentially thrown away along with the other gel from primary PCR and so, no picture was taken of the gel.
Week 10: Materials and Methods for Cloning was done. Since the first attempt at secondary PCR was a failure, another secondary PCR was done. However, the primary PCR products had been moved and the microcentrifuge tube had been bent and emptied of its products. So, primary PCR had to be done again and the results are coming in Week 11.
Week 3: The pNIC-Bsa4 culture was started. They were incubated overnight and spun down the next morning. The culture had contamination (pictures upcoming soon) of what appeared to be fungi so Aakash's pNIC culture was used for this procedure. It appeared the kanomycin was not effective for this process. The pNIC pellets (2) were placed in the -20 degree Celsius freezer for further use. Each pellet represented 40 mL worth of plasmid-containing bacterial cells.
Week 4: RE digest was done using EcoR1 and PvuII as the restriction enzymes. The inactivation temperature for PvuII was unknown and the inactivation temperature for EcoR1 was 60 degrees Celsius so 80 degrees Celsius was used for the heat block. In the 1.7 mL centrifuge tube, there was 18.95 microliters of plasmid, 2.5 microliters of 10X Enzyme buffer, 2.55 microliters of DDW to 24 microliters, 1 microliter of RE for each of the 3 tubes (the third tube had 0.5 microliters of PvuII and 0.5 microliters of EcoR1), with a total volume of 25 microliters. The gel picture is coming soon!
Later that week, Midi prep was done. Since all of the measurements in the protocol are based off of 20 mL of plasmid-containing bacterial cells and each pellet in this lab contained 40 mL of plasmid-containing bacterial cells, all the measurements were doubled from what they were in the protocol. The cells were resuspended in 1.6 mL of MX1 buffer, then 2 mL of MX2 buffer was added. 2.8 mL of chilled MX3 buffer was added, 6 mL of Wash buffer was used, and 2 mL of elution buffer was used in this lab. Speedvac was used instead of the 30-60 incubator when the membrane was dried. The elute was collected and stored for later use (Picture also coming soon!).
Week 1&2
Week 1: LB Agar solution was made.
Week 2: LB+Kan solution was made. DNA sequencing and transformation took place. The forward sequence (Figure 2) did not work as planned, with many Ns dispersed throughout. The reverse sequence (Figure 1) was much better, with fewer N nucleotides dispersed in the middle of the sequence.
Figure 1: The Reverse Sequence of DNA (top)
Figure 2: The Forward Sequence of DNA (bottom)